Hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC). Due to the inability to grow HCV in the lab, little is known about how HCV contributes to carcinogenesis. Our lab has developed a reverse genetic system for dissecting the mechanisms of RNA replication in cell culture. The proposed studies utilize this system to understand the structure and function of the HCV replication machinery, with the goal of identifying novel targets for combating HCC. 1) We have initiated genetic studies on nonstruclural proteins (NS) 3-4A, which comprise the essential viral serine protease and RNA helicase. Our results describe new and important functional interactions involving NS3 and the C-terminal acidic domain of 4A, and reveal a potential new target for drug discovery. Further biochemical and cell biological studies focus on understanding the mechanism of these interactions. 2) Genetic footprinting will be used to probe functional regions vs. regions of flexibility within the IMS genes. This technique is providing new information about the functional organization of the replicase, as well as useful tools for further characterization. 3) Little is known about the cellular determinants of HCV replication. We are examining the role of host factors and antiviral defense mechanisms in the process of HCV replication, with the aim of broadening the useful host range of the current HCV replication systems. These efforts will help to address the role of HCV in hepatocellular transformation during the development of HCC.